Effect On TNF-α Expression Of Porcine Pulmonary Alveolar Macrophages With PRRSV ADE Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) was characterized by reproductive failure in sows and gilts and respiratory diseases in piglets. PRRSV is endemic in the world's largest pork producing nations and accounts for massive global economic losses each year. Antibody-dependent enhancement of infection(ADE)is one of the most important immunological factors, and is also one of the major problems in preventing and controlling PRRS. PRRSV is known for its restricted cell tropism, primarily infecting porcine alveolar macrophages (PAM) via receptor-mediated endocytosis.TNF-α, produced mostly by macrophages, is a kind of monokines. Its main functions are inflammatory promotion and immunological regulation. Nuclear factor-κB (NF-κB), as the center of cell signal transduction, is a critical regulator of innate and adaptive immune function. NF-κB activation was characterized by translocation of NF-κB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-κB-regulated TNF-αgene expression. IKKβ, a predominant element of I-κB kinase, play a key role on NF-κB activation. In order to investigate TNF-αexpression of porcine pulmonary alveolar macrophages after PRRSV ADE infection, PAM in vitro infected with PRRSV BJ-4 strain via the ways of ADE and normal were collected and the expression of TNF-α,NF-κB and IKKβwere analyzed by Real-time PCR. The supernatant were harvested and the secretary of TNF-αwas detected.The PAM was isolated and cultured. A standard ADE assay was used to determine the susceptibility of PRRSV BJ-4 strain for ADE infection. The 104 TCID50 PRRSV was mixed with an equal volume of each of the pre-standardized, serially diluted Abs ( 2-1 to 2-8 after diluted 1:10 ). The cells were harvested at the end of the incubation period of 48 h, then the harvested virus was quantitated by virus titration using Marc-145 cells. The result indicated that 1:320 dilution of anti- PRRSV specific antibody significantly (p<0.5) enhanced the replication of PRRSV BJ-4 stain.The specific cDNA fragments of TNF-α, NF-κB, IKKβandβ-actin were amplified from PAM mRNA by RT-PCR. The purified PCR products were ligated into PMD19-T vector and then transformed into E.coli JM109. The recombinant plasmids were verified by PCR and sequencing. A series of diluted recombinant plasmids were used for SYBR Green-Ⅰr eal-time quantitative PCR. The standard curves showed high correlations between the Ct value and the concentration gradient of standard plasmid DNA. Thus, the real-time PCR for detecting TNF-α, NF-κB, IKKβandβ-actin mRNA were established.PAM was infected with PRRSV BJ-4 strain through ADE and normal way. The cells and supernatant were collected after 24 h. The mRNA transcriptions of TNF-α, NF-κB and IKKβmRNA were observed by real-time PCR. Meanwhile, protein expression of TNF-αin culture supernatant of PAM were examined by quantitative ELISA. The results showed that the mRNA transcriptions of TNF-α, NF-κB and IKKβin PAM with ADE infection were down regulated. The protein expression of TNF-αalso changed accordingly.